The present invention relates to methods for detecting extremely low amounts of botulinum neurotoxin in samples, including complex matrices like blood, plasma, and serum.
Pharmacokinetics (PK) generally is the field dedicated to the determination of the fate of substances administered externally to a living organism. The substances of interest include pharmaceutical agents, hormones, nutrients, and toxins. Pharmacokinetics includes the study of the mechanisms of absorption and distribution of an administered drug, the rate at which a drug action begins and the duration of the effect, the chemical changes of the substance in the body (e.g. by metabolic enzymes) and the effects and routes of excretion of the metabolites of the drug.
Pharmacokinetics is often studied using mass spectrometry because of the complex nature of the matrix (often plasma, serum, or urine) and the need for high sensitivity to observe concentrations after a low dose and a long time period. However, even mass spectrometry has limits on the level of detection and does not show if a biotherapeutic is active when detected. For toxins, a common test is the LD50 acute toxicity test, which tests at which amount is lethal to 50% of the animals injected with the substance within 96 hours. The test may start with eggs, embryos, or juveniles and may last from 7 to 200 days.
Due to the high potency of botulinum neurotoxin type A (BoNT/A) and the extremely low amounts of the protein being injected into patients (low nanogram therapeutic dose), no one has previously developed PK assays able to detect fully active BoNT/A circulating in patients' blood or other biological fluids. Until recently, in vitro assays for determining the potency of botulinum neurotoxin (BoNT) have not been available, and thus the only method approved by regulatory agencies for potency testing was the in vivo mouse LD50 assay. Mass Spectrometry methods to detect BoNT rely on measuring the activity of the catalytic component of BoNTs, namely the light chain, and therefore, they only detect the presence of active light chain, not the presence of fully active BoNTs. Free light chain, unable to enter neurons, will produce a positive signal in these assays. The method presented here is able to detect fully active BoNT.